CN111849467B - Infrared II-region fluorescence gold nanocluster and preparation and application thereof - Google Patents
Infrared II-region fluorescence gold nanocluster and preparation and application thereof Download PDFInfo
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Abstract
The invention relates to a preparation method and application of an infrared II-region fluorescent gold nanocluster4Dissolving in strong alkaline aqueous solution, and reducing the trivalent gold ion Au by using a reducing agent3+Reduction to gold atom Au0Selecting protein ligand template molecules containing sulfydryl (cysteine) and conjugated functional groups, and changing conditions such as temperature, reaction time and the like through a microwave-assisted synthesis device to enable gold atoms to grow and aggregate in the corresponding ligand templates to form gold nanoclusters. The gold nanocluster can emit fluorescence in an infrared II area, and has high fluorescence quantum yield and good biological safety.
Description
Technical Field
The invention relates to the technical field of infrared II-zone fluorescent materials, in particular to an infrared II-zone fluorescent gold nanocluster and preparation and application thereof.
Background
The infrared II area fluorescence imaging technology is to utilize infrared laser (b)>808nm) to emit near infrared II region fluorescence (1000nm-1700nm), and performing real-time dynamic detection at living organism level. The technology originates from an important scientific hypothesis in the research of fluorescence quantum dot biological imaging by Carroll et al in 2006: the fluorescence biological imaging effect of the near infrared II region (1000nm-1700nm) is better than that of the visible light region and the infrared I region (400-1000 nm). This assumption, once proposed, is accepted by many scholars because of its high sensitivity (detection limit: 10) compared to conventional optical imaging techniques-15mol/L), high time (<50ms) and spatial resolution: (<25 μm), deep tissue penetration ability (>2cm), low cost, no radiation, low self-luminous interference of biological tissues and the like, and becomes a hot field for biological imaging research in recent years. In 2018, the synthesis preparation of novel infrared II Materials and the imaging application of the novel infrared II Materials in organisms (such as brains, livers and intestinal tracts) are successively researched and reported in the journal of top-level engineering Materials such as Nature Nanotechnology and Nature Materials, and infrared II nano imaging becomes an exploratory and challenging hotspot research field in the biological imaging technology.
At present, researches find that the nano materials with infrared II-region fluorescence imaging performance mainly comprise the following two types: doped with rare earth element fluorescent material and heavy metal quantum dots. The two nanomaterials have different light emission mechanisms.
The rare earth element doped nano fluorescent material generally consists of a matrix and an activator, wherein the matrix is a compound (such as Y) as a material host2O3，La2O3,Gd2O3) The activator is a small amount of dopant ions (especially trivalent rare earth ions, such as: sm3+,Eu3+,Tb3+,Dy3+,Nd3+,Ho3+,Er3+,Tm3+,Yb3+) For infrared II rare earth doped nanoparticles, the emission wavelength depends on the elemental species of the doped activator. For example, Ho, Pr, Tm and Er are capable of emitting fluorescence at 1185,1310,1475 and 1525nm, respectively. Such materials have excellent optical properties such as: long fluorescence lifetime, narrow spectral line, wide fluorescence emission wavelength coverage area, etc., but low quantum yield (0.1% -3%). And the rare earth doped nano fluorescent material has poor biological stability, and rare earth elements are easily combined with phosphoric acid and converted into a rare earth phosphate compound to cause fluorescence quenching, so that the activation of inflammatory corpuscles of cells NLRP3 is triggered, and inflammatory factors are released. Therefore, the biological safety of infrared II rare earth materials is a major obstacle to clinical application.
The heavy metal quantum dot is a semiconductor nano structure which mainly comprises IV, II-VI, IV-VI or III-V group elements, contains limited number of atoms to form crystal lattices, has three dimensions of nanometer magnitude and binds excitons in three spatial directions. The light-emitting mechanism of the heavy metal quantum dot is the action of an exciton, the light-emitting process is that a negative electron of a valence band is excited to jump to a conduction band, a positive hole is left, an electron-hole combination is formed, the exciton shows fluorescence characteristics when deactivated, and simultaneously, the energy band gap between the valence band and the conduction band is changed along with the change of the lattice size of the quantum dot, so that the change of the light-emitting wavelength from visible light to infrared light is influenced. Quantum dots as a novel semiconductor nano material have many unique optical properties, especially in the infrared II fluorescence range (1000-The optical material has large absorption coefficient and high Quantum Yield (QY)>15%) and the like, and has extremely high research value in the aspect of infrared II fluorescence imaging. However, the infrared II fluorescent quantum dots are mostly composed of active heavy metals, such as: PbS, Ag2Se, CdTe/HgTe, these heavy metal elements are easily released in biological media to cause cytotoxicity. At present, researchers have proved that the heavy metal ions released by intravenous injection of the quantum dots containing the heavy metal ions have obvious toxic and side effects when acting on nerve cells.
The two types of infrared II materials have obvious cytotoxic side effects, which are just the defects and shortcomings of the prior art, so how to synthesize and prepare the infrared II area nano imaging material with good biocompatibility is an important scientific problem in the current nano biological imaging research field.
Although the gold nano material has microwave absorption performance, high surface activity, optical performance, photocatalytic property, photoelectrochemical property and photothermal property and optical conversion property (absorption, emission, scattering and plasma resonance), the gold nano material has potential application value in various biomedical imaging due to the unique physicochemical property, but the preparation and imaging application of the gold nano material in an infrared II area are not researched and realized at present. The reason is that the gold nano material has the fluorescence property depending on the particle size, and the tuning of the emission spectrum in different bands (from a visible light region to an infrared region) can be realized through the size regulation, but the gold nano material emitting light in a near-infrared region (especially 800-plus-1000 nm) does not comply with the quantum size effect theory, and the emission spectrum in a near-infrared II region is difficult to realize through the size regulation.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide an infrared II-region fluorescent gold nanocluster, and preparation and application thereof.
The first purpose of the invention is to provide a preparation method of an infrared II region fluorescent gold nanocluster, which comprises the following steps:
(1) adding an alkaline aqueous solution into a tetrachloroauric acid aqueous solution for reaction until the tetrachloroauric acid aqueous solution becomes colorless and transparent, and then adding an aqueous solution of ligand template molecules into the colorless and transparent solution to obtain a mixed solution, wherein the pH value of the mixed solution is 10-11 (preferably the pH value is 10); wherein the ligand template molecule comprises polypeptide, macromolecular compound, amino acid or protein; the ligand template molecule comprises a functional group containing sulfydryl and a conjugated molecular structure;
(2) using sodium borohydride (NaBH)4) Reduction of Au in the mixed solution with an aqueous solution3+Reduction to gold atom Au0And then continuing to react to enable the gold atoms to grow and aggregate in the ligand template molecules to form the gold nanoclusters, wherein the number of the gold atoms in the gold nanoclusters is 10-25.
Further, in the step (1), the mole ratio of the functional group containing sulfhydryl group and the functional group containing conjugated molecular structure in the ligand template molecule is 1/18-18/1.
Further, the conjugated molecule comprises one or more of imidazole ring, benzene ring and phenol.
Further, in step (1), the thiol-group-containing functional group is derived from cysteine (Cys); the functional group containing conjugated molecular structure is derived from one or more of histidine (His), tyrosine (Tyr) and tryptophan (Trp).
Further, in step (1), the molar ratio of the mercapto group-containing functional group to the conjugated molecule-containing functional group is 8: 10.
Preferably, in step (1), the ligand template molecule comprises cysteine, histidine and tyrosine. The mol ratio of cysteine, histidine and tyrosine is 5-8:6-8: 6-7.
Further, in the step (1), the protein is selected from one or more of ribonuclease (RNase-A), Bovine Serum Albumin (BSA) and beta-lactoglobulin (beta-lactoglobulin). Preferably, the protein is RNase-A.
Further, when the ligand template molecule comprises polypeptide or protein, the molar ratio of the tetrachloroauric acid to the ligand template molecule is 20-25: 1; when the ligand template molecule comprises a macromolecular compound or amino acid, the molar ratio of the tetrachloroauric acid to the ligand template molecule is 20-25: 14-18.
Further, in the step (1), the alkaline aqueous solution is an aqueous solution of sodium hydroxide (NaOH). Preferably, the concentration of the aqueous sodium hydroxide solution is 1M. In the step (1), the alkaline condition is used for deprotonating the sulfydryl of the ligand template molecule and the functional group of the conjugated molecular structure, so that the ligand molecule is more favorably combined with gold ions on the surface of the gold cluster to form a stable structure. And if the ligand template molecule and the tetrachloroauric acid mixed solution are flocculated, adding some more sodium hydroxide to ensure that the pH of the mixed solution is more than or less than the isoelectric point of the protein.
Preferably, in the step (1), the concentration of the tetrachloroauric acid aqueous solution is 15 mM; the water solution of the ligand template molecule is 10mg/mL-25 mg/mL. The concentration of the ligand template molecules increases, the fluorescence intensity of the gold cluster increases, but the emission spectrum has a blue shift of 50-200 nm.
In step (1), after addition of ligand template molecules, Au in solution3+The ions are freely distributed in the ligand template molecules to provide space for the subsequent gold atoms to aggregate to form gold nanoclusters.
Further, the molar ratio of the sodium borohydride in the step (2) to the tetrachloroauric acid in the step (1) is 0.02-0.1: 1.
Further, in the step (2), the aqueous solution of sodium borohydride is added dropwise to the mixed solution so that Au is formed3+The ions are reduced to Au0Due to Au0The high surface energy can be quickly gathered to form clusters under the microwave heating environment, and the gold ions on the surface are combined with the ligand to form a stable structure, or the reaction can be carried out for 24 to 72 hours at the temperature of 4 ℃ to ensure that Au can be formed0Slowly grow into gold clusters.
Further, in the step (2), the reduced solution is reacted at 4 ℃ for 24-72h or at 100W, 50-100 ℃ (preferably 70 ℃) for 30-90s (preferably 60s) under microwave, so that gold atoms grow and aggregate in ligand template molecules to form gold nanoclusters, and the reaction solution is finally a transparent and clear brown-black solution.
In the step (2), the sodium borohydride plays a role of a reducing agent, the addition amount of the reducing agent is controlled within a proper range, the use amount is too large, the reduction reaction is too violent, black precipitation appears in the mixed solution or the mixed solution is changed into black immediately, and a large amount of gold atoms are gathered to form gold nanoparticles instead of clusters, so that the gold nanoclusters with the infrared II area fluorescence effect are difficult to form.
Further, in step (2), after the gold nanoclusters are formed, a dialysis bag with a molecular weight cut-off of 20000Da is used to dialyze and remove unreacted ions (such as Au)3+,Na+,Cl-) The step (2).
The second purpose of the invention is to protect the infrared II-region fluorescent gold nanoclusters prepared by the preparation method, which comprises the gold nanoclusters and ligand template molecules attached to the surfaces of the gold nanoclusters, wherein the number of gold atoms in the gold nanoclusters is 10-25, and the ligand template molecules comprise polypeptides, macromolecular compounds, amino acids or proteins; and the ligand template molecule comprises a functional group containing a sulfydryl and a conjugated molecular structure.
Further, the thiol-containing functional group is derived from cysteine (Cys); the functional group containing conjugated molecular structure is derived from one or more of histidine (His), tyrosine (Tyr) and tryptophan (Trp).
Preferably, the ligand template molecule comprises a polypeptide, amino acid or protein. The polypeptide, amino acid or protein comprises amino acid containing sulfhydryl group and amino acid containing conjugated molecular structure.
Preferably, the amino acids and proteins include cysteine, histidine and tyrosine. Wherein, cysteine contains sulfydryl, histidine contains conjugated imidazole, and tyrosine contains conjugated benzene ring.
Preferably, the amino acid or protein has a molar ratio of cysteine, histidine and tyrosine of 8:4: 6.
Further, the protein is selected from one or more of ribonuclease (RNase-A), Bovine Serum Albumin (BSA) and beta-lactoglobulin.
Preferably, the ligand template molecule is selected from the group consisting of ribonuclease, beta-lactoglobulin, dihydrothio-sulfobetaine or polypeptide (CYKPCHCYKPCHYCKPYCHCKPYCHY-NH)2)。
Further, in each gold nanocluster, the total number of cysteine, histidine and tyrosine in the ligand template molecule is 14-18.
Further, the molar ratio of the tetrachloroauric acid to the ligand template molecule in step (1) is 20-25: 1.
The third purpose of the invention is to protect the application of the infrared region II fluorescent gold nanoclusters in the preparation of near-infrared region II fluorescent imaging preparations.
Further, the formulation is an oral formulation. The infrared II-region fluorescent gold nanoclusters have good biocompatibility and safety, and can enter organisms in an oral administration mode.
Further, the imaging preparation is used for detecting intestinal diseases such as mechanical ileus and obstructive intestinal cancer. The infrared II-region fluorescent gold nanocluster can effectively research the biosafety structure-activity relationship of the gold nanocluster, provide guidance for the safety design of a gold-based nano biological product, and accelerate the popularization of the gold nanocluster to clinical imaging application.
In the invention, the fluorescence gold nanocluster in the infrared II region means that the fluorescence emission peak is in the near-infrared II region, and the emission wavelength corresponding to the emission peak is 1000-1400 nm.
The infrared II-region fluorescence gold nanocluster can emit infrared II-region fluorescence, and the light emitting mechanism of the infrared II-region fluorescence gold nanocluster is caused by the combined action of two theories, namely a quantum size effect and a ligand-to-metal charge transfer theory. Influence of ligand electron donating property on metal surface gold ion (Au)1+) To internal gold atoms (Au)0) The transition energy level is excited by electrons, and the relaxation phenomenon of the electron return ground state is influenced by the cluster to the ligand energy level orbit, so that the light-emitting wavelength is changed. The invention influences the charge transfer process from a ligand in a gold cluster to metal by introducing a functional group structure containing a conjugated system and a sulfydryl, and the introduction of an aromatic conjugated system provides a wider transition range after electrons in the gold cluster are excited, so that the energy level (band gap) from the ligand to the gold cluster is further narrowed, and the luminescence red shift of the gold nanocluster to an infrared II region is further realized, and the specific principle is as follows:
when the gold nano-cluster contains gold atom number N<30 hours, the light-emitting principle basically accords with the quantum size effect, the position of an emission peak is in positive correlation with the number of gold atoms, and the emission wavelength Eev＝EFermi/N1/3(ii) a Wherein EFermiThe fermi energy of gold; n is the number of core atoms of the cluster. This theory shows that the electronic structure of the cluster depends on the free electron density of the metal as well as the cluster size. However, the infrared II region fluorescent gold nanocluster is difficult to obtain only by means of quantum size theory, and the property of the ligand must be considered.
Meanwhile, according to the charge transfer transition theory from the metal to the ligand, fluorescence is generated mainly through charge transfer of atoms in the ligand to central gold atoms, the essence of the process is the oxidation-reduction process of the metal, the transition process needs to absorb specific energy to express specific emission, and the charge transfer can occur only when the empty orbital levels of the orbital level metal of the ligand are matched. Therefore, the ligand template molecules comprise amino acid containing sulfhydryl group and amino acid containing conjugated molecule, and the electron structure on the gold cluster is greatly influenced in the combination with Au ions on the surface of the gold cluster, so that the generation of complex molecular orbitals is caused. The electron-donating property of the ligand is changed without changing the core ligand to adjust the luminous intensity and wavelength. Conjugated molecular structures contained in ligands, such as: the imidazole ring, benzene ring and phenol structure can increase the electron transition range, prolong pi bonds, enhance the electron delocalization range and enable fluorescence red shift, and the key point is that the aggregation degree of the ligand surface and the electron cloud density are changed. FIG. 1 is a schematic diagram showing the effect of different ligands on the electron transition energy level of Au; wherein FIGS. 1a1, a2 and a3 are respectively the molecular structure diagrams of combined cysteine, histidine and tyrosine with Au; FIGS. 1b1, b2, b3 are molecular orbital level diagrams after cysteine, histidine and tyrosine are combined respectively, and the forbidden bandwidths are respectively 2.308eV, 0.038eV and 0.072 eV.
By means of the scheme, the invention has the following advantages:
the invention adopts a water phase method synthesis method to synthesize the tetrachloroauric acid HAuCl4Dissolved in a strongly basic solution, andusing a reducing agent to react trivalent gold ions with Au3+Reduction to gold atom Au0Selecting a specific ligand template which comprises a sulfhydryl-containing amino acid and a functional group containing a conjugated molecule, and then growing and aggregating gold atoms in the corresponding ligand template to form the gold nanocluster by changing conditions such as temperature, reaction time and the like. The invention adopts the biological inert element Au to synthesize and prepare the infrared II nano material, can effectively improve the biocompatibility of the gold nanocluster, provides a safe and biologically-friendly novel nano material for the field of infrared II area imaging, and can regulate and control the light-emitting wavelength of the infrared II area of the gold nanocluster by regulating and controlling the quantity and the proportion of amino acid containing sulfydryl and conjugated molecules.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following description is made with reference to the preferred embodiments of the present invention and the accompanying detailed drawings.
Drawings
FIG. 1 is a schematic diagram showing the effect of different ligands on the electron transition energy level of Au;
FIG. 2 is a schematic diagram of the synthesis process of the infrared II region fluorescent gold nanoclusters;
fig. 3 is an emission spectrum of the gold nanoclusters prepared in example 1;
FIG. 4 shows the results of high resolution transmission electron microscopy and particle size distribution measurements of gold nanoclusters prepared in example 1;
FIG. 5 is atomic force microscopy characterization of gold nanoclusters prepared in example 1;
FIG. 6 is a calculated quantum yield of gold clusters in comparison to IR-26 molecules;
FIG. 7 shows MTS toxicity test results for different infrared II region nanomaterials on different cells;
FIG. 8 shows the result of living and dead staining of different cells by different IR region II nanomaterials;
fig. 9 is the mouse intestinal live imaging monitoring results of the gold nanoclusters prepared in example 1 orally taken in example 1;
description of reference numerals:
1-HAuCl4；2-NaBH4；3-RNase-A；4-Au。
Detailed Description
The following examples are given to further illustrate the embodiments of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
In the following examples of the invention, the materials used are as follows:
deionized water (resistivity 18.2m Ω cm), tetrachloroauric acid (HAuCl)4·3H2O, Au basic of not less than 49.0 percent, sodium hydroxide (NaOH), and sodium borohydride (NaBH)4,≥98.0％)，RNase-A(MW:13.7kDa,>70U/mg), BSA (purity 98%).
Example 1
(1) Dissolving ligand template molecule RNase-A in water to prepare a ligand template solution with the concentration of 10 mg/mL. In addition, tetrachloroauric acid was dissolved in water to obtain a chloroauric acid solution with a concentration of 15 mM. Both solutions were prepared in 500. mu.L.
(2) 50 μ L of 1M aqueous sodium hydroxide solution was added dropwise to the chloroauric acid solution, discoloration of the chloroauric acid solution was observed, and then a ligand template solution was added thereto to form a mixed solution. The pH of the mixed solution was about 11.
(3) After thoroughly mixing for 5 minutes, 10. mu.L of NaBH as a reducing agent was added4An aqueous solution (concentration: 15mM) was added dropwise to the above mixed solution. Putting the mixed solution in a refrigerator at 4 ℃ overnight to obtain a dark brown transparent clear mixed solution, which indicates that the reaction is finished, and the solution contains a large amount of gold nanocluster RNase-A @ AuNCs. Dissolving the product obtained in step (3) in water, dialyzing with dialysis bag with cut-off molecular weight of 20000Da to remove unreacted ions, and storing in refrigerator at 4 deg.C.
Or adding the mixed solution added with the reducing agent in the step (3) into a microwave synthesizer by using a microwave-assisted synthesis method, reacting at 70 ℃ for 60 seconds with reaction energy of 100W, and preparing the gold nanocluster, so that the finished product can be prepared in 1 minute without waiting for overnight.
Example 2
Gold nanoclusters were prepared according to the method of example 1, except that RNase-a was replaced with equimolar amounts of beta lactoglobulin, bovine Blood Serum Albumin (BSA), dihydrolipoic acid, dihydrothio-sulfobetaine, glutathione, mercaptoethanol or bioengineered polypeptides (amino acid sequence structures of which are shown in table 1).
Several gold nanoclusters prepared in the above examples were tested for their infrared II fluorescence properties, and the structures are shown in Table 1, wherein N isMercapto group/NConjugationRepresents the molar ratio of the total number of thiol groups in the ligand molecule to the total number of conjugated molecules. It can be seen from the table that eight ligand molecules can emit fluorescence in the infrared region II, and the fluorescence emission peak of the gold nanoclusters prepared from ribonuclease, β -lactoglobulin, dihydrothio-sulfobetaine, and polypeptide (containing sulfhydryl groups and conjugated molecules) customized by bioengineering is in the near infrared region II (NIR-II), while the fluorescence peak of the infrared region II of Bovine Serum Albumin (BSA), glutathione, and mercaptoethanol has a slight blue shift, because Bovine Serum Albumin (BSA), glutathione, mercaptoethanol have more sulfhydryl proportions, sulfhydryl groups will preferentially bind with Au, and under the condition of low sulfhydryl ligand proportion, conjugated molecules will tend to bind with gold clusters, so higher sulfhydryl proportion will cause blue shift of fluorescence, and higher conjugated molecules will cause red shift of the proportion fluorescence.
TABLE 1 Infrared II region fluorescence Performance test results of gold nanoclusters prepared from different ligand template molecules
In the polypeptide sequences of Table 1, H represents histidine, Y represents tyrosine, and C represents cysteine.
Fig. 3 is an emission spectrum of the gold nanoclusters prepared in example 1, which has an emission peak at 1050nm, a half-peak width of 205nm, and is in the infrared II region.
Fig. 4 is a result of a high-resolution transmission electron microscope and particle size distribution test of the gold nanoclusters prepared in example 1, and it can be seen from the figure that the gold nanoclusters are spherical, the average particle size is 2.2 ± 0.1nm, and the lattice spacing is 0.25 nm. In addition, atomic force microscopy showed the gold nanocluster particle size to be 3.5 ± 0.5nm, indicating that the gold nanocluster surface protein ligand thickness is 0.65nm (fig. 5). As can be seen in FIG. 6, the quantum yield of gold nanoclusters RNase-A @ AuNCs is 1.9%, in which IR26 is the standard control.
In addition, the gold nanoclusters prepared in example 1 were subjected to biosafety testing, and for comparison, a rare earth element-doped nano fluorescent material NaYF was selected4Er/Yb RENPs and heavy metal quantum dots Ag2S QDs were tested in the same manner.
MTS is utilized to test the toxicity of the infrared II area nano-materials with three different concentrations on HCT-116 human intestinal cancer cells, HepG-2 human liver cancer cells, THP-1 human mononuclear leucocytes and BEAS-2B human lung normal epithelial cells, and the results show that the gold nanoclusters prepared in example 1 have no toxicity basically compared with the gold of the heavy metal quantum dots and the rare earth nano-materials (figures 7-8).
The gold nanoclusters prepared in example 1 are treated by oral administration to normal mice, the abdomen of the mice needs to be unhaired, anesthesia is not needed, the oral dose of each mouse is 2mg/kg, and the gold nanoclusters are infused into the bodies of the mice by oral administration. The mouse intestinal tract was monitored by live real-time imaging, and the results are shown in fig. 9, in which fig. 9a2, b2, c2, d2, e2 and f2 correspond to enlarged views of dotted line boxes in fig. 9a1, b1, c1, d1, e1 and f1, and fig. 9a3, b3, c3, d3, e3 and f3 correspond to real-body images of isolated intestinal tracts in fig. 9a1, b1, c1, d1, e1 and f 1. The gold nanoclusters have stable infrared bifluorescent luminous capacity and can be used for detecting intestinal diseases.
Example 3
Gold nanomaterial was prepared according to the method of example 1, except that RNase-a was replaced with a small molecule amino acid: cysteine, histidine and tyrosine, and the molar ratio of the three amino acids is changed to prepare different gold nano materials.
Table 2 shows the effect of different amino acid ratios on the emission peak and morphology of the gold nanomaterials, and in table 2, NA represents no fluorescence emission. It can be seen that only a specific proportion of amino acids red-shifted the emission peak of the gold nanoclusters to the infrared II region.
Table 2 influence of different amino acid ratios on emission peak and morphology of gold nanoclusters
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Claims (7)
1. A preparation method of an infrared II-region fluorescent gold nanocluster is characterized by comprising the following steps:
(1) adding an alkaline aqueous solution into a tetrachloroauric acid aqueous solution for reaction until the tetrachloroauric acid aqueous solution becomes colorless and transparent, and then adding a ligand template molecule into the colorless and transparent solution to obtain a mixed solution, wherein the pH value of the mixed solution is 10-11; wherein the ligand template molecule is an amino acid; and the ligand template molecule comprises a functional group containing a sulfydryl group and a conjugated molecular structure;
(2) reducing Au & lt 3+ & gt in the mixed solution into gold atoms by adopting a sodium borohydride aqueous solution, and then continuing to react to ensure that the gold atoms grow and gather in ligand template molecules to form gold nanoclusters, wherein the number of the gold atoms in the gold nanoclusters is 10-25;
in step (1), the amino acids comprise cysteine, histidine and tyrosine, and the molar ratio of the cysteine, the histidine and the tyrosine is 5-8:6-8: 6-7.
2. The method of claim 1, wherein: in the step (1), the mole ratio of the functional group containing the sulfydryl in the ligand template molecule to the functional group containing the conjugated molecular structure is 1-18: 1-18.
3. The method of claim 1, wherein: in the step (1), when the ligand template molecule is amino acid, the molar ratio of the tetrachloroauric acid to the ligand template molecule is 20-25: 14-18.
4. The method of claim 1, wherein: the molar ratio of the sodium borohydride in the step (2) to the tetrachloroauric acid in the step (1) is 0.02-0.1: 1.
5. The method of claim 1, wherein: in the step (2), the reduced solution is reacted for 24-72h at 4 ℃ or reacted for 30-90s under 100W and 50-100 ℃ microwave radiation, so that gold atoms grow and aggregate in ligand template molecules to form gold nanoclusters.
6. An infrared region II fluorescent gold nanocluster prepared by the preparation method of any one of claims 1 to 5, wherein: the gold nanocluster comprises gold nanoclusters and ligand template molecules attached to the surfaces of the gold nanoclusters, wherein the number of gold atoms in the gold nanoclusters is 10-25, and the ligand template molecules are amino acids; and the ligand template molecule comprises a functional group containing a sulfydryl group and a conjugated molecular structure, the amino acid comprises cysteine, histidine and tyrosine, and the molar ratio of the cysteine to the histidine to the tyrosine is 5-8:6-8: 6-7.
7. The use of the infrared region II fluorescing gold nanoclusters of claim 6 for the preparation of near infrared region II fluorescence imaging formulations.
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